Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 2015 Mar 17;83(4):1366–1371. doi: 10.1128/IAI.02909-14

Interleukin-10 from T Cells, but Not Macrophages and Granulocytes, Is Required for Chronic Disease in Leishmania mexicana Infection

Laurence U Buxbaum 1,✉
Editor: J A Appleton
PMCID: PMC4363430  PMID: 25605773

Abstract

Chronic cutaneous disease of mice caused by the protozoan parasite Leishmania mexicana requires interleukin-10 (IL-10) and FcγRIII (an activating IgG receptor). Macrophages readily secrete IL-10 in response to IgG-coated amastigotes, making macrophages a prime candidate as the critical source of IL-10. However, indirect evidence suggested that macrophage IL-10 is not essential for chronic disease. I now show directly that mice lacking IL-10 from macrophages and granulocytes still have chronic disease, like wild-type C57BL/6 mice. However, T cell-derived IL-10 is required for chronic disease. CD4-cre IL-10flox/flox mice lack IL-10 from T cells (both CD4+ and CD8+) and heal their L. mexicana lesions, with parasite control. I had previously shown that depletion of CD25+ T cells had no effect on chronic disease, and thus, T cells other than CD25+ regulatory T (Treg) cells should be the important source of IL-10. Given that conventional T cells do not express FcγRs, there is likely to be an indirect pathway by which FcγRIII on some other cell engaged by IgG1-amastigote immune complexes induces IL-10 from T cells. Further work is needed to delineate these pathways.

INTRODUCTION

The protozoan parasite Leishmania causes ∼2 million new cases a year, with 12 million individuals infected at any given time, and is a leading cause of death worldwide from parasite infections, second only to malaria (1). Leishmania mexicana causes cutaneous and diffuse cutaneous leishmaniasis, and disease is generally chronic in humans and in C57BL/6 (B6) mice, unlike Leishmania major infection, where humans and B6 mice control disease and parasite numbers (2, 3). Understanding how the parasite suppresses the host immune response is critical for solutions to this important human disease.

The classical Th1/Th2 immunologic paradigm does not adequately explain the immune response to Leishmania species that cause chronic New World cutaneous leishmaniasis (L. mexicana complex parasites, such as L. mexicana, L. amazonensis, and L. pifanoi) (4). This is not too surprising, as L. mexicana is separated from L. major by 40 million to 80 million years of evolution (4). For example, although interleukin-12 (IL-12) is the master regulator of Th1 responses, IL-12-deficient mice show no phenotype and display chronic disease similar to that of wild-type (WT) B6 mice when infected with L. mexicana (5). This is because IL-10 suppresses the IL-12 pathway (6). We previously demonstrated a critical role for IL-10 in suppressing a protective immune response, as IL-10-deficient B6 mice heal their lesions and control parasite numbers effectively, unlike WT B6 mice, which develop nonhealing chronic disease (6).

Lesions in L. mexicana infection contain large numbers of macrophages and neutrophils but few lymphocytes, which is different from L. major infection, where lymphocytes are more prevalent (5). Furthermore, we have shown a critical role for FcγRIII in chronic disease caused by L. mexicana, with FcγRIII-deficient B6 mice clearing parasites and resolving lesions, similarly to IL-10-deficient mice (7). Macrophages are known to express a variety of receptors for IgG (FcγR), including FcγRIII, and secrete IL-10 in response to lipopolysaccharide (LPS) and immune complexes consisting of IgG-coated Leishmania amastigotes (6, 8). Conventional T cells, on the other hand, do not express FcγRs (9). Taken together, these data imply that macrophages are an excellent candidate as the crucial source of IL-10 responsible for chronic disease.

Macrophages express both FcγRIII, which binds IgG1 immune complexes, and FcγRI, which binds IgG2a/c and its immune complexes. We found previously that macrophages secrete IL-10 equally well in response to L. mexicana amastigotes coated with IgG1 and IgG2a/c serum antibodies (10). However, IgG1-deficient mice develop stronger and earlier IgG2a/c responses to L. mexicana surface epitopes and are resistant to infection (10). Thus, in vivo, IgG1, but not IgG2a/c, is associated with suppression of the protective Th1 response. This implies that macrophages may not actually be the critical source of IL-10.

To resolve this question, I used tissue-specific IL-10 knockout (KO) mice. I found that mice lacking macrophage and granulocyte IL-10 (Lyz2-cre/cre IL-10fl/fl mice) did not fully control L. mexicana infection and had chronic disease and immune responses similar to those of B6 mice. In contrast, mice lacking IL-10 from CD4+ and CD8+ T cells (CD4-cre IL-10fl/fl mice) healed their lesions and controlled parasites similarly to IL-10-deficient mice. Therefore, T cells but not macrophages or granulocytes are an important source of IL-10 that suppresses a protective immune response to L. mexicana infection.

MATERIALS AND METHODS

Mice.

Female C57BL/6 (B6) and B6 Lyz2-cre [B6.129P2-Lyz2tm1(cre)Ifo/J] mice were purchased from Jackson Laboratories (Bar Harbor, ME). B6 IL-10fl/fl mice were a generous gift of Yuri Rubtsov and the original producers, Axel Roers and Werner Müller (11). B6 Lyz2-cre mice were bred to B6 IL-10fl/fl mice to generate Lyz2-cre/cre IL-10fl/fl mice (double homozygotes). B6 CD4-cre [B6.Cg-Tg(CD4-cre)1Cwi N9] mice were purchased from Taconic Farms (Germantown, NY) and bred to B6 IL-10fl/fl mice to generate B6 CD4-cre IL-10fl/fl mice. All mice used in these experiments are on a B6 background and were backcrossed to B6 mice for >10 generations. Genotypes were determined by PCR of tail snips. Courses of infection were carried out with groups of at least 5 mice per experiment. Female mice were purchased at 4 to 10 weeks of age and were age matched for all experiments. Animals were maintained in a specific-pathogen-free environment, and the animal colony was screened regularly, and tested negative, for the presence of murine pathogens. Studies were reviewed and approved by the IACUC, Biosafety, and R&D Committees of the VA Medical Center of Philadelphia.

Parasites and antigens.

L. mexicana (MNYC/BZ/62/M379) promastigotes were grown in Grace's medium as previously described (5). Stationary-phase promastigotes (day 7 of culture) were washed three times in Dulbecco's modified Eagle's medium (DMEM), and 5 × 106 parasites (in 50 μl DMEM) were injected into the hind footpad of mice. Lesions were monitored by using a metric dial caliper, and lesion size was defined as the footpad thickness of the infected foot minus the footpad thickness of the contralateral uninfected foot. Axenic amastigotes, grown free of mammalian cells, were prepared by placing L. mexicana stationary-phase promastigote cultures (day 7) at 33°C for at least 3 days. Freeze-thaw antigen (FTAg) was prepared from L. mexicana stationary-phase promastigotes that were washed four times in phosphate-buffered saline (PBS), resuspended at 109 parasites/ml, and frozen (−80°C) and thawed rapidly (37°C) for five cycles. FTAg was assayed for protein content by the BCA (bicinchoninic acid) method (Pierce, Rockford, IL), brought to 1 mg/ml protein, aliquoted, and stored at −80°C. “Washed membranes” were prepared from axenic amastigotes by hypotonic lysis, as described previously (7).

Cytokine assays.

Single-cell suspensions were prepared from draining lymph nodes (LNs), and 200-μl samples (8 × 105 cells) were cultured in duplicate in 96-well tissue culture plates in DMEM (Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 25 mM HEPES (pH 7.4), 50 μM 2-mercaptoethanol (2-ME), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were stimulated with 10 μg/ml (∼107 cell equivalents/ml) L. mexicana FTAg, 1 μg/ml plate-bound anti-CD3 (BD Biosciences, San Diego, CA), or medium alone for 3 days at 37°C in a 5% CO2 incubator, and previously frozen supernatants were assayed by an enzyme-lined immunosorbent assay (ELISA) for gamma interferon (IFN-γ) and IL-4 as previously described (12) and for IL-10 by using commercial antibodies as recommended by the manufacturer (BD Bioscience, San Diego, CA). Cells from uninfected mice had no detectable IL-10, IL-4, or IFN-γ production with antigen stimulation in these experiments. Macrophage supernatants were assayed for IL-10 by an ELISA in the same manner.

In vitro infection of bone marrow-derived macrophages.

Bone marrow-derived macrophages were prepared and infected at a ratio of 10 IgG-opsonized L. mexicana axenic amastigotes per cell, as described previously (13). LPS from Escherichia coli O111:B4 (Sigma-Aldrich, MO) was added at 100 ng/ml. Purified anti-IL-10R (1B1.3a, a generous gift from DNAX) was added to cultures at 9 mg/ml. Supernatants from cultures grown for 20 h were assayed for IL-10 by an ELISA, as described above.

Measurement of Leishmania-specific serum IgG.

Sera from infected mice were assayed for Leishmania-specific IgG1 and IgG2a/c by an ELISA using washed membranes from axenic amastigotes for capture and biotin-conjugated anti-mouse IgG1 and IgG2a/c (BD Biosciences) with peroxidase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA). C57BL/6 mice actually produce IgG2c rather than IgG2a. IgG levels are shown as means and standard errors of the means for 5 mice per group. Relative quantitation was performed as described previously (10), using serum from L. mexicana-infected B6 mice as the standard.

Parasite quantitation.

Parasite quantitation was performed by limiting dilution, as described previously, for 5 mice per group (14). The limit of detection was 1.4 logs, equal to 25 parasites/lesion.

Statistics.

Macrophage cultures were incubated in quadruplicate, and means and standard errors are shown. All experiments were performed at least twice, and representative data are shown. Groups of at least 4 to 5 mice were used for all experiments. A Student t test was used to compare groups of mice, or replicate samples, with significance set at a P value of ≤0.05.

Ethics.

Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Philadelphia Veterans Affairs Medical Center (Office of Laboratory Welfare permit number A3076-01) and were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Academy of Sciences (15) as well as all other U.S. government federal guidelines.

RESULTS

Macrophages from Lyz2-cre/cre IL-10fl/fl mice do not secrete IL-10 in response to immune complexes and LPS.

Macrophages from C57BL/6 mice secrete IL-10 when stimulated with LPS and immune complexes (8). Lyz2-cre/cre IL-10fl/fl mice have been analyzed and lack IL-10 production from macrophages and granulocytes due to the specific expression of the lysozyme (Lyz2) promoter in these cells (16). I confirmed that B6, but not Lyz2-cre/cre IL-10fl/fl, macrophages secrete IL-10 in response to L. mexicana amastigote immune complexes and LPS (Fig. 1).

FIG 1.

FIG 1

Open in a new tab

Macrophages from Lyz2-cre/cre IL-10fl/fl mice do not secrete IL-10 in response to immune complexes and LPS. Bone marrow-derived macrophages were prepared from C57BL/6 (B6) and Lyz2-cre/cre IL-10fl/fl mice and were stimulated for 20 h with medium alone (media), LPS, unopsonized L. mexicana axenic amastigotes and LPS (aa+LPS), and L. mexicana axenic amastigotes opsonized with serum from infected mice and LPS (IgG-ops aa+LPS). Supernatants were assayed for IL-10. Data are from two experiments with identical results.

Lyz2-cre/cre IL-10fl/fl mice have chronic disease with L. mexicana infection.

To directly assess whether IL-10 from macrophages is important for chronic disease seen with L. mexicana infection, I compared infections of WT B6 mice and Lyz2-cre/cre IL-10fl/fl mice. I found no difference in the course of infection (Fig. 2A), with both mouse strains having chronic and nonprogressive disease. In addition, parasite loads did not differ between these mice (Fig. 2B).

FIG 2.

FIG 2

Open in a new tab

Lyz2-cre/cre IL-10fl/fl mice have chronic disease with L. mexicana infection similar to that of B6 mice. (A) Groups of 10 B6 and Lyz2-cre/cre IL-10fl/fl mice were infected in the hind footpad with 5 × 106 L. mexicana stationary promastigotes, and lesion size was monitored. (B) Parasite loads were determined by limiting dilution at 18 weeks of infection. (C) At 18 weeks of infection, draining LN cells were stimulated with L. mexicana FTAg, and IFN-γ was assayed by an ELISA after 3 days of stimulation. (D) At 20 weeks of infection, draining LN cells were stimulated with L. mexicana FTAg, and IL-10 was assayed by an ELISA after 3 days of stimulation. (E) Sera from Lyz2-cre/cre IL-10fl/fl mice infected for 18 weeks with L. mexicana were assayed for anti-L. mexicana IgG1 and IgG2a/c that bind to washed membranes at various dilutions (dil). Data are representative of six experiments with similar results.

I next analyzed the immune response from antigen-induced recall responses of LN cells from infected mice. There was no statistically significant difference between the IFN-γ responses of WT B6 and Lyz2-cre/cre IL-10fl/fl mice in six independent experiments, with some experiments showing somewhat more IFN-γ from WT mice and some showing more from Lyz2-cre/cre IL-10fl/fl mice but with no statistically significant difference in any experiment, even when 10 mice per group were used (P = 0.2) (Fig. 2C). I cannot rule out small differences in IFN-γ levels between WT and Lyz2-cre/cre IL-10fl/fl mice. In addition, IL-10 responses from draining LN cells were not significantly different (Fig. 2D), nor were antigen-specific IgG1 and IgG2c responses to parasite antigens (Fig. 2E). In multiple experiments, levels of IL-4 responses were low in both Lyz2-cre/cre IL-10fl/fl and WT B6 mice and never statistically different (data not shown).

CD4-cre/IL-10fl/fl mice are resistant to L. mexicana infection.

Given that macrophage-derived IL-10 is not critical for chronic disease caused by L. mexicana infection, I next examined whether IL-10 from T cells is important. Because CD4 is expressed in the double-positive (CD4+ CD8+) stage of thymic development, mice expressing cre recombinase under the control of the CD4 promoter (17) delete the IL-10 gene from nearly all mature T cells, including CD4+ and CD8+ T cells. I infected CD4-cre IL-10fl/fl mice with L. mexicana and found that, unlike B6 controls, these mice heal their lesions (Fig. 3A).

FIG 3.

FIG 3

Open in a new tab

CD4-cre IL-10fl/fl mice are resistant to L. mexicana infection. (A) Groups of five B6 and CD4-cre IL-10fl/fl mice were infected in the hind footpad and monitored as described in the legend of Fig. 2 (*, P < 0.05). (B) Parasite loads were determined by limiting dilution at various times postinfection (*, P < 0.02). (C to E) At 5 weeks (C), 8 weeks (D), and 20 weeks (E) of infection, draining LN cells were stimulated with medium alone (media), plate-bound anti-CD3, and L. mexicana FTAg, and supernatants were assayed for IFN-γ after 3 days of stimulation by an ELISA (*, P < 0.05; #, P < 0.002). (F) The same supernatants from cells stimulated with FTAg, shown in panels C to E, were also assayed for IL-10 by an ELISA (*, P = 0.02; #, P < 0.001). These data are representative of three experiments with similar results.

Although identical at 4 weeks of infection, by 8 weeks of infection, before lesion healing began, CD4-cre/IL-10fl/fl mice had significantly fewer parasites than did B6 mice (1,700-fold) (Fig. 3B). This difference increased steadily to 350,000-fold by 21 weeks of infection (Fig. 3B).

At 5 weeks postinfection, before parasite loads had diminished, recall responses from draining LN cells of CD4-cre IL-10fl/fl mice showed stronger IFN-γ production induced by parasite antigen stimulation with freeze-thawed antigen (FTAg) (2.4-fold) and anti-CD3 (4.1-fold) (Fig. 3C). At 8 weeks of infection, when parasite loads had already begun to drop in CD4-cre IL-10fl/fl mice, antigen-induced recall responses of draining LN cells revealed that IFN-γ responses were no longer different in CD4-cre IL-10fl/fl and B6 mice. However, the level of anti-CD3-induced IFN-γ was still higher in CD4-cre IL-10fl/fl mice than in B6 mice (Fig. 3D). By 20 weeks of infection, when antigen loads had diminished markedly (as indicated by very large differences in parasite loads), CD4-cre IL-10fl/fl mice actually showed a trend toward weaker IFN-γ responses than those of B6 mice (Fig. 3E). However, IFN-γ responses were far weaker by this time point. In multiple experiments, IL-4 responses in both CD4-cre IL-10fl/fl and WT B6 mice were weak and never statistically different (data not shown).

We showed previously that in L. mexicana infection of B6 mice, IL-10 from draining LN cells comes from T cells (92%) (7). I therefore examined whether T cell-specific IL-10-deficient mice have less IL-10 from the LN recall response. In fact, IL-10 production was significantly diminished in CD4-cre/IL-10fl/fl mice compared to B6 controls at 5 weeks (2.4×), 8 weeks (3.0×), and 20 weeks (11.4×) of infection (Fig. 3F).

Macrophages from CD4-cre IL-10fl/fl mice have no defect in IL-10 secretion in response to immune complexes and LPS.

I wanted to be certain that the Il10 gene deletion in T cells did not affect macrophage secretion of IL-10, as some supposed tissue-specific gene deletions are not completely specific. For example, DNA prepared from tail snips of lck-cre IL-10fl/fl mice, which should have shown Il10 gene deletion in T cells only, frequently had no detectible IL-10 genes, demonstrating nonspecific Il10 gene deletion (data not shown). In fact, I found that CD4-cre IL-10fl/fl bone marrow-derived macrophages had no defect in the ability to secrete IL-10 in response to L. mexicana immune complexes and LPS (Fig. 4).

FIG 4.

FIG 4

Open in a new tab

Macrophages from CD4-cre IL-10fl/fl mice have no defect in IL-10 secretion in response to immune complexes and LPS. Bone marrow-derived macrophages were prepared from B6 mice and CD4-cre IL-10fl/fl mice and were stimulated for 20 h with medium alone (media), LPS, unopsonized L. mexicana axenic amastigotes and LPS (aa+LPS), and L. mexicana axenic amastigotes opsonized with serum from infected mice and LPS (IgG-ops aa+LPS). Supernatants were assayed for IL-10. Data are representative of three experiments with similar results.

Late, but not early, in infection, CD4-cre IL-10fl/fl mice have weaker IgG1 and IgG2c responses than those of B6 mice.

Parasite-specific serum IgG levels were assayed at 8 and 21 weeks of infection. At 8 weeks, IgG1 levels were the same in L. mexicana-infected B6 and CD4-cre IL-10fl/fl mice (Fig. 5A). IgG2c levels were low at 8 weeks but also not distinguishable between WT and CD4-cre IL-10fl/fl mice. By 21 weeks of infection, CD4-cre IL-10fl/fl mice had lower serum levels of parasite-specific IgG1 and IgG2c than did B6 mice (Fig. 5B). Because ELISA optical density (OD) values do not give a linear response, I calculated relative amounts of antigen-specific IgG and found that the IgG1 level was 4.7-fold higher and the IgG2c level was 9.8-fold higher in B6 than in CD4-cre IL-10fl/fl mice (Fig. 5C).

FIG 5.

FIG 5

Open in a new tab

Late, but not early, in infection, CD4-cre IL-10fl/fl mice have weaker IgG1 and IgG2a/c responses than those of B6 mice. (A and B) Groups of five B6 and CD4-cre IL-10fl/fl mice were infected in the hind footpad as described in the legend of Fig. 2, and sera were obtained at 8 weeks (A) and 21 weeks (B) of infection. Sera were assayed for IgG1 and IgG2a/c that bind to washed membranes from L. mexicana axenic amastigotes. (C) Relative IgG responses for serum samples assayed at a 1/40 dilution were calculated for IgG1 and IgG2a/c at 21 weeks (*, P ≤ 0.05 for OD values at a 1/40 dilution and for log fold changes for relative IgG levels). Values were normalized to B6 levels for each isotype. Data are representative of three experiments with similar results.

DISCUSSION

Macrophages predominate in the lesions of L. mexicana-infected B6 mice. The importance of IgG1 bound to the surface of amastigotes and of FcγRIII has already been demonstrated in vivo (10). However, because macrophages express FcγRI as well as FcγRIII and secrete IL-10 in response to IgG1 and IgG2a/c, there was circumstantial evidence that cells other than macrophages might be responsible for the critical IL-10 production that suppresses a needed Th1 response. In addition, IL-10 from T cells has important roles in other Leishmania infections (18–22). I have now presented direct data showing that T cell IL-10, but not macrophage (or granulocyte) IL-10, is required for chronic disease in murine L. mexicana infection. A model of the role of IL-10 and various cells and cytokines is shown (Fig. 6A).

FIG 6.

FIG 6

Open in a new tab

Model of the immune response against Leishmania mexicana and the role of IL-10. (A) Killing of Leishmania requires IFN-γ, primarily from CD4+ Th1 cells, and inducible nitric oxide synthase in infected macrophages (MΦ). IL-10 has pleiotropic effects on the immune response. It is known to downregulate MHC class II and the costimulatory proteins B7-1 and B7-2 on antigen-presenting cells such as macrophages and dendritic cells. It also downregulates proinflammatory cytokines such as IL-12, which is critical for T cell development into IFN-γ-producing Th1 cells. In addition, IL-10 downregulates the expression of inducible nitric oxide synthase, which is required for nitric oxide production, which in turn is required for Leishmania killing. The immunosuppressive pathway requires IL-10 but also FcγRIII and IgG1. Thus, IL-10 reduces productive Th1 development and IFN-γ production, as well as directly downregulating inducible nitric oxide synthase. I have now shown that IL-10 from macrophages is dispensable for the immunosuppressive pathway. TCR, T cell receptor. (Modified from reference 7.) (B) In the indirect model, IgG1-Leishmania immune complexes bind to FcγRIII on an unknown cell type, inducing cell surface receptors or soluble factors that induce IL-10 production from T cells. This FcγRIII+ cell should lack FcγRI and FcγRIV. In the direct model, a T cell subset expresses FcγRIII and secretes IL-10 in response to IgG1-Leishmania immune complexes. Other signals, such as TLR ligands, may also be required for optimal IL-10 production from T cells.

A role for IL-10 from T cells but not macrophages/granulocytes in L. major infection of B6 mice was recently shown, although the effect is small and found only early in infection (22). Furthermore, it is clear that L. major and L. mexicana have very different immunologic responses, with a clear Th1/Th2 dichotomy in L. major infection but not in L. mexicana infection. In addition, lymphocytic infiltration of lesions is much more predominant in L. major infection. We previously showed that IL-10 from lesions but not IL-10 from lymph node cells correlates with a healing phenotype (7). This makes the importance of IL-10 from T cells that much more surprising. The fact that macrophages can produce IL-10 in response to parasite immune complexes (at least in vitro) and yet are not sufficient or necessary for chronic disease in this system suggests either an in vivo blockade of this pathway (perhaps due to a lack of Toll-like receptor [TLR] signaling) or that close proximity of the IL-10-producing T cells providing a cell surface marker and/or cytokine is also required. Similar findings have been reported for chronic L. major Seidman infection of B6 mice, in which innate cells produce IL-10, which is not important, whereas IL-10 from T cells is important (21).

The course of infection of CD4-cre IL-10fl/fl mice is similar to that seen in IL-10 knockout mice (6). Lesions grow faster in the absence of IL-10; thus, early IL-10 diminishes the inflammatory response in the lesion, due to either cell influx or proliferation. However, parasite burdens early on (first 4 weeks) are not greatly affected by this early IL-10 production.

As we have shown previously, IgG2a/c is not fully driven by IFN-γ, as FcγRIII KO mice produce more IFN-γ with L. mexicana infection than do WT mice but have identical IgG2c responses (10). CD4-cre IL-10fl/fl mice have stronger IFN-γ responses, but IgG2c levels are lower than those in B6 mice. Rather, IgG1 and IgG2c responses are attenuated in CD4-cre IL-10fl/fl mice, indicating that T cell IL-10 may enhance IgG responses (both Th1 and Th2) and that its lack suppresses this effect. It has been reported that IL-10, when combined with TLR stimulation, increases IgG secretion in human B cells in vitro through the TLR-MyD88-STAT3 pathway (23) or through a CD40 pathway (24, 25). In vivo, it was found that blocking IL-10 in mice diminished dehydroepiandrosterone-induced IgG production (26). These data, taken together with my data, support a role for IL-10 enhancement of IgG responses.

The finding that T cells are the critical source of IL-10 in chronic L. mexicana infection is now clear but is puzzling. Because conventional T cells do not express FcγRs, there are two possibilities. A direct pathway would require a T cell to express FcγRIII (to bind IgG1 immune complexes) but not FcγRI or FcγRIV (thus not binding IgG2a/c immune complexes) (Fig. 6B). NKT cells express FcγRIII, but I have observations that do not support their role in chronic L. mexicana disease (data not shown). γδ-T cells can also express FcγRIII (27) but are not major histocompatibility complex (MHC) restricted and generally are CD4− CD8−, with a few cells expressing CD8αα (28), and thus should not delete the Il10 gene in CD4-cre IL-10fl/fl mice. Dendritic cells (DCs) generally express FcγRI and FcγRIII and produce IL-10 in response to both IgG2c and IgG1 parasite immune complexes in vitro (data not shown). Alternatively, FcγRIII+ cells could induce IL-10 from a T cell by some combination of cytokines and cell surface interactions. This would be an indirect pathway (Fig. 6B) and may in fact have multiple intervening steps. These cells are not likely to be macrophages due to macrophage expression of FcγRI (and clear responsiveness to IgG2a/c immune complexes) as well as FcγRIII (with responsiveness to IgG1 immune complexes), given that both FcγRs signal through the same common γ chain. Even though previous macrophage FcγR work focused on IL-10 production, other downstream effects should also proceed through the common γ chain in all activating FcγRs. Some DCs express IL-4 (29), so a role for DCs as an important source of IL-10 is still possible. However, I have some preliminary data that bone marrow DCs also respond to IgG1 and IgG2a/c equally well (data not shown) and are not good candidates for the FcγRIII+ cell, although I cannot completely rule out some DC subset in this role. Some mast cell types can express FcγRIII (30). This area requires further research to better understand this pathway.

We previously found that in the draining lymph nodes of B6 mice infected with L. mexicana, nearly all IL-10 is produced by T cells, and the vast majority of IL-10 from CD4+ T cells came from CD25+ CD4+ cells (a population that includes regulatory T [Treg] cells) (7). However, depletion of CD25+ cells did not alter the course of infection. Also, LN IL-10 patterns were not different in healing FcγRIII KO mice and B6 mice with chronic disease. However, the IL-10 levels in lesions were different in these two mouse strains. Unfortunately, cells from footpad lesions are not easily assessed. In L. major infection, IL-10 from CD25+ Foxp3+ Treg cells and IL-10 from CD4+ CD25− Foxp3− IFN-γ+ cells each play a role (19, 21, 31). Whether L. mexicana infection has similar CD4+ CD25− Foxp3− T cells has not yet been determined.

ACKNOWLEDGMENTS

I thank Niansheng Chu, Aaron Deleonguerrero, Ben Armon, and Magdalene Moy for technical help with this project.

This material is the result of work supported with resources and the use of facilities at the Philadelphia Veterans Affairs Medical Center. The work was funded by National Institutes of Health NIAID grant R01AI081717, a Department of Veterans Affairs merit review, and the University of Pennsylvania.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

REFERENCES

  • 1.WHO. 2005. Leishmaniasis. World Health Organization, Geneva, Switzerland. http://www.who.int/leishmaniasis/en/ Accessed 2 May 2014. [Google Scholar]
  • 2.Preston PM, DuMonde DC. 1976. Immunology of clinical and experimental leishmaniasis, p 168–202. In Cohen S, Sadun EH (ed), Immunology of parasitic infections. Blackwell Scientific Publications, Oxford, United Kingdom. [Google Scholar]
  • 3.Alexander J, Blackwell JM. 1986. The immunological significance of genetically determined cross reactivity between taxonomically distinct Leishmania species, p 185–191. In Rioux JA (ed), Leishmania. IMEEE, Montpellier, France. [Google Scholar]
  • 4.McMahon-Pratt D, Alexander J. 2004. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunol Rev 201:206–224. doi: 10.1111/j.0105-2896.2004.00190.x. [DOI] [PubMed] [Google Scholar]
  • 5.Buxbaum LU, Uzonna JE, Goldschmidt MH, Scott P. 2002. Control of New World cutaneous leishmaniasis is interleukin-12 independent but STAT4 dependent. Eur J Immunol 32:3206–3215. doi:. [DOI] [PubMed] [Google Scholar]
  • 6.Buxbaum LU, Scott P. 2005. Interleukin 10- and Fcgamma receptor-deficient mice resolve Leishmania mexicana lesions. Infect Immun 73:2101–2108. doi: 10.1128/IAI.73.4.2101-2108.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Thomas BN, Buxbaum LU. 2008. FcgammaRIII mediates immunoglobulin G-induced interleukin-10 and is required for chronic Leishmania mexicana lesions. Infect Immun 76:623–631. doi: 10.1128/IAI.00316-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kane MM, Mosser DM. 2001. The role of IL-10 in promoting disease progression in leishmaniasis. J Immunol 166:1141–1147. doi: 10.4049/jimmunol.166.2.1141. [DOI] [PubMed] [Google Scholar]
  • 9.Ravetch JV, Bolland S. 2001. IgG Fc receptors. Annu Rev Immunol 19:275–290. doi: 10.1146/annurev.immunol.19.1.275. [DOI] [PubMed] [Google Scholar]
  • 10.Chu N, Thomas BN, Patel SR, Buxbaum LU. 2010. IgG1 is pathogenic in Leishmania mexicana infection. J Immunol 185:6939–6946. doi: 10.4049/jimmunol.1002484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Roers A, Siewe L, Strittmatter E, Deckert M, Schluter D, Stenzel W, Gruber AD, Krieg T, Rajewsky K, Muller W. 2004. T cell-specific inactivation of the interleukin 10 gene in mice results in enhanced T cell responses but normal innate responses to lipopolysaccharide or skin irritation. J Exp Med 200:1289–1297. doi: 10.1084/jem.20041789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Scharton-Kersten T, Afonso LC, Wysocka M, Trinchieri G, Scott P. 1995. IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J Immunol 154:5320–5330. [PubMed] [Google Scholar]
  • 13.Buxbaum L. 2013. Leishmania mexicana infection induces IgG to parasite surface glycoinositol phospholipids that can induce IL-10 in mice and humans. PLoS Negl Trop Dis 7:e2224. doi: 10.1371/journal.pntd.0002224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Buxbaum LU, Denise H, Coombs GH, Alexander J, Mottram JC, Scott P. 2003. Cysteine protease B of Leishmania mexicana inhibits host Th1 responses and protective immunity. J Immunol 171:3711–3717. doi: 10.4049/jimmunol.171.7.3711. [DOI] [PubMed] [Google Scholar]
  • 15.National Research Council. 2011. Guide for the care and use of laboratory animals, 8th ed. National Academies Press, Washington, DC. [Google Scholar]
  • 16.Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. 1999. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res 8:265–277. doi: 10.1023/A:1008942828960. [DOI] [PubMed] [Google Scholar]
  • 17.Lee PP, Fitzpatrick DR, Beard C, Jessup HK, Lehar S, Makar KW, Perez-Melgosa M, Sweetser MT, Schlissel MS, Nguyen S, Cherry SR, Tsai JH, Tucker SM, Weaver WM, Kelso A, Jaenisch R, Wilson CB. 2001. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 15:763–774. doi: 10.1016/S1074-7613(01)00227-8. [DOI] [PubMed] [Google Scholar]
  • 18.Holaday BJ, Pompeu MM, Jeronimo S, Texeira MJ, de A Sousa A, Vasconcelos AW, Pearson RD, Abrams JS, Locksley RM. 1993. Potential role for interleukin-10 in the immunosuppression associated with kala azar. J Clin Invest 92:2626–2632. doi: 10.1172/JCI116878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Belkaid Y, Hoffmann KF, Mendez S, Kamhawi S, Udey MC, Wynn TA, Sacks DL. 2001. The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J Exp Med 194:1497–1506. doi: 10.1084/jem.194.10.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Stober CB, Lange UG, Roberts MT, Alcami A, Blackwell JM. 2005. IL-10 from regulatory T cells determines vaccine efficacy in murine Leishmania major infection. J Immunol 175:2517–2524. doi: 10.4049/jimmunol.175.4.2517. [DOI] [PubMed] [Google Scholar]
  • 21.Anderson CF, Oukka M, Kuchroo VJ, Sacks D. 2007. CD4+CD25-Foxp3- Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis. J Exp Med 204:285–297. doi: 10.1084/jem.20061886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Schwarz T, Remer K, Nahrendorf W, Masic A, Siewe L, Muller W, Roers A, Moll H. 2013. T cell-derived IL-10 determines leishmaniasis disease outcome and is suppressed by a dendritic cell based vaccine. PLoS Pathog 9:e1003476. doi: 10.1371/journal.ppat.1003476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Liu BS, Stoop JN, Huizinga TW, Toes RE. 2013. IL-21 enhances the activity of the TLR-MyD88-STAT3 pathway but not the classical TLR-MyD88-NF-kappaB pathway in human B cells to boost antibody production. J Immunol 191:4086–4094. doi: 10.4049/jimmunol.1300765. [DOI] [PubMed] [Google Scholar]
  • 24.Rousset F, Garcia E, Defrance T, Peronne C, Vezzio N, Hsu DH, Kastelein R, Moore KW, Banchereau J. 1992. Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc Natl Acad Sci U S A 89:1890–1893. doi: 10.1073/pnas.89.5.1890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Burdin N, Rousset F, Banchereau J. 1997. B-cell-derived IL-10: production and function. Methods 11:98–111. doi: 10.1006/meth.1996.0393. [DOI] [PubMed] [Google Scholar]
  • 26.Cheng GF, Tseng J. 2000. Regulation of murine interleukin-10 production by dehydroepiandrosterone. J Interferon Cytokine Res 20:471–478. doi: 10.1089/10799900050023889. [DOI] [PubMed] [Google Scholar]
  • 27.Braakman E, van de Winkel JG, van Krimpen BA, Jansze M, Bolhuis RL. 1992. CD16 on human gamma delta T lymphocytes: expression, function, and specificity for mouse IgG isotypes. Cell Immunol 143:97–107. doi: 10.1016/0008-8749(92)90008-D. [DOI] [PubMed] [Google Scholar]
  • 28.Carding SR, Egan PJ. 2002. Gammadelta T cells: functional plasticity and heterogeneity. Nat Rev Immunol 2:336–345. doi: 10.1038/nri797. [DOI] [PubMed] [Google Scholar]
  • 29.Vremec D, Pooley J, Hochrein H, Wu L, Shortman K. 2000. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol 164:2978–2986. doi: 10.4049/jimmunol.164.6.2978. [DOI] [PubMed] [Google Scholar]
  • 30.Chong HJ, Andrew Bouton L, Bailey DP, Wright H, Ramirez C, Gharse A, Oskeritzian C, Xia HZ, Zhu J, Paul WE, Kepley C, Schwartz LB, Ryan JJ. 2003. IL-4 selectively enhances FcgammaRIII expression and signaling on mouse mast cells. Cell Immunol 224:65–73. doi: 10.1016/j.cellimm.2003.08.004. [DOI] [PubMed] [Google Scholar]
  • 31.Anderson CF, Mendez S, Sacks DL. 2005. Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice. J Immunol 174:2934–2941. doi: 10.4049/jimmunol.174.5.2934. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES